Multiplex MRM Assay for Evaluation of Cancer

ABSTRACT

The current disclosure provides specific peptides, and derived ionization characteristics of the peptides from the estrogen receptor (ER), progesterone receptor (PR), and/or antigen Ki67 (Ki67) proteins that are particularly advantageous for quantifying the ER, PR, and/or Ki67 proteins directly in biological samples that have been fixed in formalin by the method of Selected Reaction Monitoring/Multiple Reaction Monitoring (SRM/MRM) mass spectrometry. Such biological samples are chemically preserved and fixed wherein the biological sample is selected from tissues and cells treated with formaldehyde containing agents/fixatives including formalin-fixed tissue/cells, formalin-fixed/paraffin embedded (FFPE) tissue/cells, FFPE tissue blocks and cells from those blocks, and tissue culture cells that have been formalin fixed and or paraffin embedded. A protein sample is prepared from a biological sample using the Liquid Tissue™ reagents and protocol, and the ER, PR, and/or Ki67 proteins are quantitated in the Liquid Tissue™ sample by the method of SRM/MRM mass spectrometry by quantitating in the protein sample at least one or more of the peptides described for one or more of the ER, PR, and/or Ki67 proteins. These peptides can be quantitated if they reside in a modified or in an unmodified form. An example of a modified form of an ER, PR, and/or Ki67 peptide is phosphorylation of a tyrosine, threonine, serine, and/or other amino acid residues within the peptide sequence.

This application claims priority to U.S. Application Ser. No.61/537,918, filed Sep. 22, 2011, entitled “Multiplex MRM Assay forEvaluation of Cancer,” the contents of which are hereby incorporated byreference in their entirety.

INTRODUCTION

Specific peptides derived from subsequences of the estrogen receptor(ER), the progesterone receptor (PR), and the antigen Ki67 (Ki67)proteins are provided. The peptide sequences andfragmentation/transition ions for each peptide are particularly usefulin a mass spectrometry-based Selected Reaction Monitoring (SRM), whichalso can be referred to as a Multiple Reaction Monitoring (MRM) assay.Such assays are alternatively referred to herein as SRM/MRM. Informationabout the use (individually, simultaneously, or in various combinations)of peptides for SRM/MRM quantitative analysis of these proteins isdescribed.

Results from the SRM/MRM assays can be used to measure relative orabsolute quantitative levels of one or more of the specific peptidesfrom (ER), the progesterone receptor (PR), and the antigen Ki67 (Ki67)proteins. Where the proteins are analyzed individually, simultaneously,or in various combinations the assays can be used to correlate accurateand precise quantitative levels of the proteins within specific breasttissue samples (e.g., cancer tissue sample), or any other tissue oforigin other than breast, of the patient or subject from whom the tissue(biological sample) was collected and preserved. This not only providesdiagnostic information about the breast cancer, but also permits aphysician or other medical professional to determine appropriate therapyfor the breast cancer patient. Such an assay that providesdiagnostically and therapeutically important information about levels ofprotein expression in a diseased tissue or other patient sample istermed a “companion diagnostic” assay. For example, such an assay can bedesigned to diagnose the stage or degree of a breast cancer, or anyother cancer, and determine a therapeutic agent to which a patient ismost likely to respond.

SUMMARY

The assays described herein are assays for measuring relative orabsolute levels of specific unmodified peptides from the ER, PR, and theKi67 proteins. Also, described herein are assays for measuring absoluteor relative levels of specific modified peptides from the ER, PR, andKi67 proteins. Examples of modifications include phosphorylated aminoacid residues (e.g. phosphotyrosine, phosphoserine and phosphothreonine)and glycosylated amino acid residues (e.g. glycosylated asparagineresidues) that are present on the peptides.

Relative quantitative levels of the ER, PR, and Ki67 proteins aredetermined by the SRM/MRM methodology, for example, by comparing SRM/MRMsignature peak areas (for example, signature peak area or integratedfragment ion intensity) of an individual ER, PR, and/or Ki67 peptide indifferent samples. Alternatively, it is possible to compare multipleSRM/MRM signature peak areas for multiple ER, PR, and/or Ki67 signaturepeptides, where each peptide has its own specific SRM/MRM signaturepeak, to determine the relative ER, PR, and/or Ki67 protein content inone biological sample with the ER, PR, and/or Ki67 protein content inone or more additional or different biological samples. In this way, theamount (or level) of a particular peptide, or peptides, from the ER, PR,and/or Ki67 proteins, and therefore the amount of the ER, PR, and/orKi67 proteins, is determined relative to the same ER, PR, and/or Ki67peptide, or peptides, across 2 or more biological samples under the sameexperimental conditions. In addition, relative quantitation can bedetermined for a given peptide, or peptides, from the ER, PR, and/orKi67 proteins within a single sample by comparing the signature peakarea for that peptide by SRM/MRM methodology to the signature peak areafor another and different peptide, or peptides, from a differentprotein, or proteins, within the same protein preparation from thebiological sample. In this way, the amount of a particular peptide fromthe ER, PR, and/or Ki67 proteins, and therefore the amount of the ER,PR, and/or Ki67 protein, is determined relative one to another withinthe same sample. These approaches generate quantitation of an individualpeptide, or peptides, from the ER, PR, and/or Ki67 proteins to theamount of another peptide, or peptides, between samples and withinsamples wherein the amounts as determined by peak area are relative oneto another, regardless of the absolute weight to volume or weight toweight amounts of the ER, PR, and/or Ki67 peptide, or peptides, in theprotein preparation from the biological sample. Relative quantitativedata about individual signature peak areas between different samples arenormalized to the amount of protein analyzed per sample. Relativequantitation can be performed across many peptides from multipleproteins and the ER, PR, and/or Ki67 proteins individually orsimultaneously in a single sample and/or across many samples to gaininsight into relative protein amounts, one peptide/protein with respectto other peptides/proteins.

Absolute quantitative levels of the ER, PR, and/or Ki67 proteins aredetermined by, for example, the SRM/MRM methodology whereby the SRM/MRMsignature peak area of an individual peptide from the ER, PR, and/orKi67 proteins in one biological sample is compared to the SRM/MRMsignature peak area of an exogenously added “spiked” internal standard.In one embodiment, the internal standard is a synthetic version of thesame exact ER, PR, and/or Ki67 peptide that contains one or more aminoacid residues labeled with one or more heavy isotopes. Suitable isotopelabeled internal standards are synthesized so that when analyzed by massspectrometry it generates a predictable and consistent SRM/MRM signaturepeak that is different and distinct from the native ER, PR, and/or Ki67peptide signature peak and which can be used as a comparator peak. Thus,when the internal standard is spiked in a known amount into a proteinpreparation from a biological sample and analyzed by mass spectrometry,the SRM/MRM signature peak area of the native peptide from the samplecan be compared to the SRM/MRM signature peak area of the internalstandard peptide. This numerical comparison provides either the absolutemolarity and/or absolute weight of the native peptide present in theoriginal protein preparation from the biological sample. Absolutequantitative data for fragment peptides are displayed according to theamount of protein analyzed per sample. Absolute quantitation can beperformed across many peptides, and thus proteins, simultaneously in asingle sample and/or across many samples to gain insight into absoluteprotein amounts in individual biological samples and in entire cohortsof individual samples.

The SRM/MRM assay method can be used to aid diagnosis of the stage ofcancer, for example, directly in patient-derived tissue, such asformalin fixed tissue, and to aid in determining which therapeutic agentwould be most advantageous for use in treating that patient. Cancertissue, including breast cancer tissue, that is removed from a patienteither through surgery, such as for therapeutic removal of partial orentire tumors, or through biopsy procedures conducted to determine thepresence or absence of suspected disease, is analyzed to determinewhether or not a specific protein, or proteins, and which forms ofproteins, are present in that patient tissue. Moreover, the expressionlevel of an individual protein such as ER, or multiple proteins such asER, PR, and/or Ki67 can be determined, individually or in a simultaneousfashion, and compared to a “normal” or reference level for each proteinor collections of proteins found in healthy tissue. Normal or referencelevels of proteins found in healthy tissue may be derived from, forexample, the relevant tissues of one or more individuals that do nothave cancer. Alternatively, normal or reference levels may be obtainedfor individuals with cancer by analysis of relevant tissues not affectedby the cancer.

Assays of protein levels (e.g., ER, PR, and/or Ki67 levels) can also beused to diagnose the stage of cancer, including breast cancer, in apatient or subject diagnosed with cancer by employing the ER, PR, and/orKi67 levels. Levels or amounts of proteins or peptides can be defined asthe quantity expressed in moles, mass or weight of a protein or peptidedetermined by the SRM/MRM assay. The level or amount may be normalizedto total the level or amount of protein or another component in thelysate analyzed (e.g., expressed in micromoles/microgram of protein ormicrograms/microgram of protein). In addition, the level or amount of aprotein or peptide may be determined on volume basis, expressed, forexample, in micromolar or nanograms/microliter. The level or amount ofprotein or peptide as determined by the SRM/MRM assay can also benormalized to the number of cells analyzed. Information regarding ER,PR, and/or Ki67 can thus be used to aid in determining stage or grade ofa cancer by correlating the level of the ER, PR, and/or Ki67 proteins(or fragment peptides of the ER, PR, and/or Ki67 proteins) with levelsobserved in normal tissues.

Once the stage and/or grade, and/or ER, PR, and/or Ki67 proteinexpression characteristics of the cancer, including breast cancer, hasbeen determined, that information can be matched to a list oftherapeutic agents (chemical and biological) developed to specificallytreat cancer tissue that is characterized by, for example, abnormalexpression of the protein or protein(s) (e.g., ER, PR, and/or Ki67) thatwere assayed. Matching information from an ER, PR, and/or Ki67 proteinassay to a list of therapeutic agents that specifically targets, forexample, the ER, PR, and/or Ki67 protein or cells/tissue expressing theprotein, defines what has been termed a personalized medicine approachto treating disease. The assay methods described herein form thefoundation of a personalized medicine approach by using analysis ofproteins from the patient's own tissue as a source for diagnostic andtreatment decisions. These proteins (ER, PR, and/or Ki67) can be usedindividually, doubly, or all three (3) simultaneously in a personalizedmedicine approach to not only breast cancer but any other cancer aswell. This collection of proteins is most advantageously applied tobreast cancer diagnosis and treatment.

These and other aspects of the present disclosure will become apparentto the skilled person in view of the description set forth below.\

DETAILED DESCRIPTION

The Selected Reaction Monitoring/Multiple Reaction Monitoring (SRM/MRM)assay can be used to measure relative or absolute quantitative levels ofone or more of the specific peptides from the ER, PR, and/or Ki67proteins, individually, in combinations, or simultaneously, andtherefore provide a means of measuring the amount of the ER, PR, and/orKi67 proteins in a given protein preparation obtained from a biologicalsample by mass spectrometry.

More specifically, the SRM/MRM assay can measure these peptides directlyin complex protein lysate samples prepared from cells procured frompatient tissue samples, such as formalin fixed cancer patient tissue.Methods of preparing protein samples from formalin fixed tissue aredescribed in U.S. Pat. No. 7,473,532, the contents of which are herebyincorporated by references in their entireties. The methods described inU.S. Pat. No. 7,473,532 may conveniently be carried out using LiquidTissue™ reagents and protocol available from Expression Pathology Inc.(Rockville, Md.).

The most widely and advantageously available form of tissues from cancerpatients tissue is formalin fixed, paraffin embedded tissue.Formaldehyde/formalin fixation of surgically removed tissue is by farand away the most common method of preserving cancer tissue samplesworldwide and is the accepted convention for standard pathologypractice. Aqueous solutions of formaldehyde are referred to as formalin.“100%” formalin consists of a saturated solution of formaldehyde (thisis about 40% by volume or 37% by mass) in water, with a small amount ofstabilizer, usually methanol to limit oxidation and degree ofpolymerization. The most common way in which tissue is preserved is tosoak whole tissue for extended periods of time (8 hours to 48 hours) inaqueous formaldehyde, commonly termed 10% neutral buffered formalin,followed by embedding the fixed whole tissue in paraffin wax for longterm storage at room temperature. Thus molecular analytical methods toanalyze formalin fixed cancer tissue will be the most accepted andheavily utilized methods for analysis of cancer patient tissue.

In principle, any predicted peptide derived from the ER, PR, and/or Ki67proteins, prepared for example by digesting with a protease of knownspecificity (e.g. trypsin), can be used as a surrogate reporter todetermine the abundance of ER, PR, and/or Ki67 proteins in a sampleusing a mass spectrometry-based SRM/MRM assay. Similarly, any predictedpeptide sequence containing an amino acid residue at a site that isknown to be potentially modified in the ER, PR, and/or Ki67 proteinsalso might potentially be used to assay the extent of modification ofthe ER, PR, and/or Ki67 proteins in a sample.

According to one embodiment, ER, PR, and/or Ki67 fragment peptides maybe generated in a variety of ways including using Liquid Tissue™protocol described, for example, in U.S. Pat. No. 7,473,532. LiquidTissue™ protocol and reagents produce peptide samples suitable for massspectroscopic analysis from formalin fixed paraffin embedded tissue byproteolytic digestion of the proteins in the tissue/biological sample.Suitable reagents and protocols also are commercially available fromOncoPlexDx (formerly Expression Pathology Inc., Rockville, Md.).

In the Liquid Tissue™ protocol the tissue/biological is heated in abuffer for an extended period of time (e.g., from about 80° C. to about100° C. for a period of time from about 10 minutes to about 4 hours) toreverse or release protein cross-linking. The buffer employed is aneutral buffer, (e.g., a Tris-based buffer, or a buffer containing adetergent). Following heat treatment the tissue/biological sample istreated with one or more proteases, including but not limited totrypsin, chymotrypsin, pepsin, and endoproteinase Lys-C for a timesufficient to disrupt the tissue and cellular structure of thebiological sample and to liquefy the sample. Exemplary conditions forthe protease treatment are from 30 minutes to 24 hours at a temperaturefrom 37° C. to 65° C.). Advantageously, endoproteases, and particularlycombinations of two or three endoproteases, used either simultaneouslyor sequentially, are employed to liquefy the sample. For example,suitable combinations of proteases can include, but are not limited to,combinations of trypsin, endoproteinase Lys-C and chemotrypsin, such astrypsin and endoproteinase Lys-C. The result of the heating andproteolysis is a liquid, soluble, dilutable biomolecule lysate.Advantageously, this liquid lysate is free of solid or particulatematter that can be separated from the lysate by centrifugation.

Surprisingly, it was found that many potential peptide sequences fromthe ER, PR, and/or Ki67 proteins are unsuitable or ineffective for usein mass spectrometry-based SRM/MRM assays for reasons that are notimmediately evident. As it was not possible to predict the most suitablepeptides for MRM/SRM assay, it was necessary to experimentally identifymodified and unmodified peptides in actual Liquid Tissue™ lysates todevelop a reliable and accurate SRM/MRM assay for the ER, PR, and/orKi67 proteins. While not wishing to be bound by any theory, it isbelieved that some peptides might, for example, be difficult to detectby mass spectrometry because they do not ionize well or producefragments that are not distinct from those generated from otherproteins. Peptides may also fail to resolve well in separation (e.g.,liquid chromatography), or may adhere to glass or plastic ware.

ER, PR, and/or Ki67 peptides found in various embodiments of thisdisclosure (e.g., Tables 1 and 2 below) were derived from the ER, PR,and/or Ki67 proteins by protease digestion of all the proteins within acomplex Liquid Tissue™ lysate prepared from cells procured from formalinfixed cancer tissue. Unless noted otherwise, in each instance theprotease was trypsin. The Liquid Tissue™ lysate was then analyzed bymass spectrometry to determine those peptides derived from the ER, PR,and/or Ki67 proteins that are detected and analyzed by massspectrometry. Identification of a specific preferred subset of peptidesfor mass-spectrometric analysis is based on: 1) experimentaldetermination of which peptide or peptides from a protein ionize in massspectrometry analyses of Liquid Tissue™ lysates, and 2) the ability ofthe peptide to survive the protocol and experimental conditions used inpreparing a Liquid Tissue™ lysate. This latter property extends not onlyto the amino acid sequence of the peptide but also to the ability of amodified amino acid residue within a peptide to survive in modified formduring the sample preparation.

Protein lysates from cells procured directly from formalin(formaldehyde) fixed tissue were prepared using the Liquid Tissue™reagents and protocol. This entails collecting cells into a sample tubevia tissue microdissection followed by heating the cells in the LiquidTissue™ buffer for an extended period of time. Once the formalin-inducedcross linking has been negatively affected, the tissue/cells are thendigested to completion in a predictable manner using a protease, suchas, trypsin. The skilled artisan will recognize that other proteases,and in particular, endoproteases may be used in place of, or in additionto, trypsin. Each protein lysate was used to prepare a collection ofpeptides by digestion of intact polypeptides with the protease orprotease combination. Each protein lysate is used to prepare acollection of peptides by digestion of intact polypeptides with theprotease or protease combination. Each Liquid Tissue™ lysate wasanalyzed (e.g., by ion trap mass spectrometry) to perform multipleglobal proteomic surveys of the peptides where the data was presented asidentification of as many peptides as could be identified by massspectrometry from all cellular proteins present in each protein lysate.An ion trap mass spectrometer or another form of a mass spectrometerthat is capable of performing global profiling for identification of asmany peptides as possible from a single complex protein/peptide lysateis employed. Ion trap mass spectrometers may, however, be the best typeof mass spectrometer for conducting global profiling of peptides.Although SRM/MRM assays can be developed and performed on any type ofmass spectrometer, including a MALDI, ion trap, or triple quadrupole isan advantageous instrument platform for SRM/MRM assays is oftenconsidered to be a triple quadrupole instrument platform.

Once as many peptides as possible were identified in a single massspectrometric analysis of a single lysate under the conditions employed,then the list of identified peptides was collated and used to determinethe proteins that were detected in that lysate. This process wasrepeated for multiple Liquid Tissue™ lysates, and the very large list ofpeptides was collated into a single dataset. The resulting datasetrepresents the peptides that can be detected in the type of biologicalsample that was analyzed (after protease digestion), and specifically ina Liquid Tissue™ lysate of the biological sample, and thus includes thepeptides for specific proteins, such as for example the ER, PR, and/orKi67 proteins.

In one embodiment, the ER, PR, and/or Ki67 tryptic peptides identifiedas useful in the determination of absolute or relative amounts of theER, PR, and/or Ki67 proteins include one or more, two or more, three ormore, four or more, five or more, six or more, eight or more, or nine ormore of the peptides of SEQ ID NO:1, SEQ ID NO:2, SEQ ID NO:3, SEQ IDNO:4, SEQ ID NO:5, SEQ ID NO:6, SEQ ID NO:7, SEQ ID NO:8, SEQ ID NO:9and/or SEQ ID NO:10, each of which are listed in Table 1. Each of thosepeptides was detected by mass spectrometry in Liquid Tissue™ lysatesprepared from formalin fixed, paraffin embedded tissue. Thus, each ofthe peptides in Table 1, or any combination of those peptides (e.g., oneor more, two or more, three or more, four or more, five or more, six ormore, eight or more, or ten or more of those peptides recited in Table1, and particularly combinations with the peptides found in Table 2) arecandidates for use in quantitative SRM/MRM assay for the ER, PR, and/orKi67 proteins in human biological samples, including directly informalin fixed patient tissue, and more specifically in formalin fixedbreast cancer patient tissue. Table 2 shows additional informationregarding some of the peptides shown in Table 1.

TABLE 1 SEQ ID Peptide Sequence SEQ ID NO: 1Leu Ala Gln Leu Leu Leu Ile Leu Ser His Ile Arg SEQ ID NO: 2Leu Leu Phe Ala Pro Asn Leu Leu Leu Asp Arg SEQ ID NO: 3Ala Gly Leu Thr Leu Gln Gln Gln His Gln Arg SEQ ID NO: 4Val Leu Leu Leu Leu Asn Thr Ile Pro Leu Glu Gly Leu Arg SEQ ID NO: 5Val Gly Asp Ser Ser Gly Thr Ala Ala Ala His Lys SEQ ID NO: 6Leu Asp Leu Thr Glu Asn Leu Thr Gly Ser Lys SEQ ID NO: 7Val Glu Pro Val Gly Asp Val Val Ser Thr Arg SEQ ID NO: 8Ala Ala Asn Leu Trp Pro Ser Pro Leu Met Ile Lys SEQ ID NO: 9Asn Val Val Pro Leu Tyr Asp Leu Leu Leu Glu Met Leu Asp Ala His ArgSEQ ID NO: 10 Ser Leu Pro Gly Phe Arg

TABLE 2 Mono Precursor Peptide Isotopic Charge Precursor Transition IonSEQ ID sequence Mass State m/z m/z Type SEQ ID NO: 1 LAQLLLILSHIR1388.89 2 695.451  738.462 y6 2 695.451  851.546 y7 2 695.451  964.63 Y82 695.451 1077.714 y9 SEQ ID NO: 2 LLFAPNLLLDR 1283.76 2 642.887 840.493 y7 2 642.887  911.53 Y8 2 642.887 1058.599 y9 SEQ ID NO: 3AGLTLQQQHQR 1278.68 2 640.347  696.353 y5 2 640.347  824.412 y6 2640.347  937.496 y7 2 640.347 1038.543 y8 SEQ ID NO: 4 VLLLLNTIPLEGLR1562.98 2 782.495  684.403 y6 2 782.495  898.535 y8 2 782.495 1012.578y9 2 782.495 1125.662 y10 SEQ ID NO: 5 VGDSSGTAAAHK 1099.53 2 550.77 598.33 y6 2 550.77  655.352 y7 2 550.77  742.384 y8 2 550.77  829.416y9 2 550.77  944.443 y10 SEQ ID NO: 6 LDLTENLTGSK 1189.62 2 595.817 619.34 y6 2 595.817  748.383 y7 2 595.817  849.431 y8 2 595.817 962.515 y9 SEQ ID NO: 7 VEPVGDVVSTR 1156.61 2 579.312  561.335 y5 2579.312  733.383 y7 2 579.312  832.452 y8 2 579.312  929.505 y9

The ER, PR, and/or Ki67 tryptic peptides listed in Table 1 include thosedetected from multiple Liquid Tissue™ lysates of multiple differentformalin fixed tissues of different human organs including prostate,colon, and breast. Each of those peptides is considered useful forquantitative SRM/MRM assay of the ER, PR, and/or Ki67 proteins informalin fixed tissue. Further data analysis of these experimentsindicated no preference is observed for any specific peptides from anyspecific organ site. Thus, each of these peptides is believed to besuitable for conducting SRM/MRM assays of the ER, PR, and/or Ki67proteins on a Liquid Tissue™ lysate from any formalin fixed tissueoriginating from any biological sample or from any organ site in thebody.

One consideration for conducting an SRM/MRM assay is the type ofinstrument that may be employed in the analysis of the peptides.Although SRM/MRM assays can be developed and performed on any type ofmass spectrometer, including a MALDI, ion trap, or triple quadrupole,the most advantageous instrument platform for SRM/MRM assay is oftenconsidered to be a triple quadrupole instrument platform. That type of amass spectrometer may be considered to be the most suitable instrumentfor analyzing a single isolated target peptide within a very complexprotein lysate that may consist of hundreds of thousands to millions ofindividual peptides from all the proteins contained within a cell.

In order to most efficiently implement an SRM/MRM assay for each peptidederived from the ER, PR, and/or Ki67 proteins it is desirable to utilizeinformation in addition to the peptide sequence in the analysis. Thatadditional information may be used in directing and instructing the massspectrometer (e.g. a triple quadrupole mass spectrometer), to performthe correct and focused analysis of specific targeted peptide(s), suchthat the assay may be effectively performed.

The additional information about target peptides in general, and aboutspecific ER, PR, and/or Ki67 peptides, may include one or more of themono isotopic mass of the peptide, its precursor charge state, theprecursor m/z value, the m/z transition ions, and the ion type of eachtransition ion. Table 2 shows additional information for some peptidesshown in Table 1 that may be used to develop an SRM/MRM assay for theER, PR, and/or Ki67 proteins.

The methods described below can be used to: 1) identify candidatepeptides from the ER, PR, and/or Ki67 proteins that can be used for amass spectrometry-based SRM/MRM assay for the ER, PR, and/or Ki67proteins, 2) develop individual SRM/MRM assays, or multiplexed assays,for target peptides from the ER, PR, and/or Ki67 proteins in order tocorrelate to breast cancer, and 3) apply quantitative assays to breastcancer diagnosis and/or choice of optimal therapy for breast cancer, andany other cancer analyzed by described SRM/MRM assays.

Assay Methods

I. Identification of SRM/MRM Candidate Fragment Peptides for the ER, PR,and/or Ki67 Proteins:

-   -   a. Prepare a Liquid Tissue™ protein lysate from a formalin fixed        biological sample using a protease or proteases, (that may or        may not include trypsin), to digest proteins    -   b. Analyze all protein fragments in the Liquid Tissue™ lysate on        an ion trap tandem mass spectrometer and identify all fragment        peptides from the ER, PR, and/or Ki67 proteins, where individual        fragment peptides do not contain any peptide modifications such        as phosphorylations or glycosylations    -   c. Analyze all protein fragments in the Liquid Tissue™ lysate on        an ion trap tandem mass spectrometer and identify all fragment        peptides from the ER, PR, and/or Ki67 proteins that carry        peptide modifications such as for example phosphorylated or        glycosylated residues    -   d. All peptides generated by a specific digestion method from        the entire, full length ER, PR, and/or Ki67 proteins potentially        can be measured, but preferred peptides used for development of        the SRM/MRM assay are those that are identified by mass        spectrometry directly in a complex Liquid Tissue™ protein lysate        prepared from a formalin fixed biological sample    -   e. Peptides that are specifically modified (phosphorylated,        glycosylated, etc.) in patient tissue and which ionize, and thus        detected, in a mass spectrometer when analyzing a Liquid Tissue™        lysate from a formalin fixed biological sample are identified as        candidate peptides for assaying peptide modifications of the ER,        PR, and/or Ki67 proteins        II. Mass Spectrometry Assay for Fragment Peptides from ER, PR,        and/or Ki67 Proteins    -   a. SRM/MRM assay on a triple quadrupole mass spectrometer for        individual fragment peptides identified in a Liquid Tissue™        lysate is applied to peptides from the ER, PR, and/or Ki67        proteins        -   i. Determine optimal retention time for a fragment peptide            for optimal chromatography conditions including but not            limited to gel electrophoresis, liquid chromatography,            capillary electrophoresis, nano-reversed phase liquid            chromatography, high performance liquid chromatography, or            reverse phase high performance liquid chromatography        -   ii. Determine the mono isotopic mass of the peptide, the            precursor charge state for each peptide, the precursor m/z            value for each peptide, the m/z transition ions for each            peptide, and the ion type of each transition ion for each            fragment peptide in order to develop an SRM/MRM assay for            each peptide.        -   iii. SRM/MRM assay can then be conducted using the            information from (i) and (ii) on a triple quadrupole mass            spectrometer where each peptide has a characteristic and            unique SRM/MRM signature peak that precisely defines the            unique SRM/MRM assay as performed on a triple quadrupole            mass spectrometer    -   b. Perform SRM/MRM analysis so that the amount of the fragment        peptide of the ER, PR, and/or Ki67 proteins that is detected, as        a function of the unique SRM/MRM signature peak area from an        SRM/MRM mass spectrometry analysis, can indicate both the        relative and absolute amount of the ER, PR, and/or Ki67 proteins        in a particular protein lysate.        -   i. Relative quantitation may be achieved by:            -   1. Determining increased or decreased presence of the                ER, PR, and/or Ki67 proteins by comparing the SRM/MRM                signature peak area from a given ER, PR, and/or Ki67                peptide detected in a Liquid Tissue™ lysate from one                formalin fixed biological sample to the same SRM/MRM                signature peak area of the same ER, PR, and/or Ki67                fragment peptide in at least a second, third, fourth or                more Liquid Tissue™ lysates from least a second, third,                fourth or more formalin fixed biological samples.            -   2. Determining increased or decreased presence of the                ER, PR, and/or Ki67 proteins by comparing the SRM/MRM                signature peak area from a given ER, PR, and/or Ki67                peptide detected in a Liquid Tissue™ lysate from one                formalin fixed biological sample to SRM/MRM signature                peak areas developed from fragment peptides from other                proteins, in other samples derived from different and                separate biological sources, where the SRM/MRM signature                peak area comparison between the 2 samples for a peptide                fragment are normalized to amount of protein analyzed in                each sample.            -   3. Determining increased or decreased presence of the                ER, PR, and/or Ki67 proteins by comparing the SRM/MRM                signature peak area for a given ER, PR, and/or Ki67                peptide to the SRM/MRM signature peak areas from other                fragment peptides derived from different proteins within                the same Liquid Tissue™ lysate from the formalin fixed                biological sample in order to normalize changing levels                of ER, PR, and/or Ki67 proteins to levels of other                proteins that do not change their levels of expression                under various cellular conditions.            -   4. These assays can be applied to both unmodified                fragment peptides and for modified fragment peptides of                the ER, PR, and/or Ki67 proteins, where the                modifications include but are not limited to                phosphorylation and/or glycosylation, and where the                relative levels of modified peptides are determined in                the same manner as determining relative amounts of                unmodified peptides.        -   ii. Absolute quantitation of a given peptide may be achieved            by comparing the SRM/MRM signature peak area for a given            fragment peptide from the ER, PR, and/or Ki67 proteins in an            individual biological sample to the SRM/MRM signature peak            area of an internal fragment peptide standard spiked into            the protein lysate from the biological sample.            -   1. The internal standard is a labeled synthetic version                of the fragment peptide from the ER, PR, and/or Ki67                proteins that is being interrogated. This standard is                spiked into a sample in known amounts, and the SRM/MRM                signature peak area can be determined for both the                internal fragment peptide standard and the native                fragment peptide in the biological sample separately,                followed by comparison of both peak areas.            -   2. This can be applied to unmodified fragment peptides                and modified fragment peptides, where the modifications                include but are not limited to phosphorylation and/or                glycosylation, and where the absolute levels of modified                peptides can be determined in the same manner as                determining absolute levels of unmodified peptides.

III. Apply Fragment Peptide Quantitation to Cancer Diagnosis andTreatment

-   -   a. Perform relative and/or absolute quantitation of fragment        peptide levels of the ER, PR, and/or Ki67 proteins and        demonstrate that the previously-determined association, as well        understood in the field of cancer, of ER, PR, and/or Ki67        protein expression to the stage/grade/status of cancer in        patient tumor tissue is confirmed.    -   b. Perform relative and/or absolute quantitation of fragment        peptide levels of the ER, PR, and/or Ki67 proteins individually,        in combinations, or all simultaneously, and demonstrate        correlation with clinical outcomes from different treatment        strategies, wherein this correlation has already been        demonstrated in the field or can be demonstrated in the future        through correlation studies across cohorts of patients and        tissue from those patients. Once either previously established        correlations or correlations derived in the future are confirmed        by this assay then the assay method can be used to determine        optimal treatment strategy.

The information shown in Table 2 is desirable to develop an SRM/MRMassay for quantitation of the ER, PR, and/or Ki67 proteins on atriplequadrupole mass spectrometer. Specific and unique characteristicsabout these ER, PR, and/or Ki67 peptides were developed by analysis ofall ER, PR, and/or Ki67 peptides on an ion trap and/or triple quadrupolemass spectrometers. That information includes the monoisotopic mass ofthe peptide, its precursor charge state, the precursor m/z value, thetransition m/z values of the precursor, and the ion types of each of theidentified transitions. That information must be determinedexperimentally for each and every candidate SRM/MRM peptide directly inLiquid Tissue™ lysates from formalin fixed tissue; because,interestingly, not all peptides from the ER, PR, and/or Ki67 proteinscan be detected in such lysates using SRM/MRM as described herein,indicating that ER, PR, and/or Ki67 peptides not detected cannot beconsidered candidate peptides for developing an SRM/MRM assay for use inquantitating peptides/proteins directly in Liquid Tissue™ lysates fromformalin fixed tissue.

Utilizing this information, quantitative SRM/MRM assays can be developedfor the ER, PR, and/or Ki67 proteins, and assessment of ER, PR, and/orKi67 protein levels in tissues based on analysis of formalin fixedbreast cancer patient-derived tissue can provide diagnostic, prognostic,and therapeutically-relevant information about each particular breastcancer patient and/or cancer patient that has a different cancer that isnot breast cancer.

In one embodiment, this disclosure describes a method for measuring thelevel of the ER, PR, and/or Ki67 proteins in a biological sample,comprising detecting and/or quantifying the amount of one or moremodified or unmodified ER, PR, and/or Ki67 fragment peptides in aprotein digest prepared from the biological sample using massspectrometry; and calculating the level of modified or unmodified ER,PR, and/or Ki67 proteins in the sample; and wherein the level is arelative level or an absolute level. In a related embodiment, thisdisclosure provides a method for quantifying one or more ER, PR, and/orKi67 fragment peptides, wherein the method comprises determining theamount of one or more of the ER, PR, and/or Ki67 fragment peptides in abiological sample by comparison to an added internal standard peptide ofknown amount, wherein each of the ER, PR, and/or Ki67 fragment peptidesin the biological sample is compared to an internal standard peptidehaving the same amino acid sequence. In some embodiments the internalstandard is an isotopically labeled internal standard peptide comprisingone or more heavy stable isotopes selected from ¹⁸O, ¹⁷O, ³⁴S, ¹⁵N, ¹³C,²H or combinations thereof.

The methods for measuring levels of the ER, PR, and/or Ki67 proteins ina biological sample described herein (or fragment peptides as surrogatesthereof) are useful as diagnostic indicators of cancer in a patient orsubject. In one embodiment, the results from the measurements of levelsof the ER, PR, and/or Ki67 protein may be employed to determine thediagnostic stage/grade/status of a breast cancer, or another cancer thatis not of breast origin, by correlating (e.g., comparing) the level ofER, PR, and/or Ki67 proteins found in a tissue with the level of theseproteins found in normal and/or cancerous or precancerous tissues.

Embodiments

-   1. A method for measuring the amount of the ER, PR, and/or Ki67    proteins in a biological sample, comprising detecting and/or    quantifying the amount of one or more modified or unmodified ER, PR,    and/or Ki67 fragment peptides in a protein digest prepared from the    biological sample using mass spectrometry; and calculating the    amount of modified or unmodified ER, PR, and/or Ki67 proteins in the    sample; and wherein the amount is a relative amount or an absolute    amount.-   2. The method of embodiment 1, further comprising the step of    fractionating the protein digest prior to detecting and/or    quantifying the amount of one or more modified or unmodified ER, PR,    and/or Ki67 fragment peptides.-   3. The method of embodiment 2, wherein the fractionating step is    selected from the group consisting of gel electrophoresis, liquid    chromatography, capillary electrophoresis, nano-reversed phase    liquid chromatography, high performance liquid chromatography, and    reverse phase high performance liquid chromatography.-   4. The method of any of embodiments 1 to 3, wherein the protein    digest of the biological sample is prepared by the Liquid Tissue™    protocol.-   5. The method of any of embodiments 1 to 3, wherein the protein    digest comprises a protease digest.-   6. The method of embodiment 5, wherein the protein digest comprises    a trypsin digest.-   7. The method of any of embodiments 1 to 6, wherein the mass    spectrometry comprises tandem mass spectrometry, ion trap mass    spectrometry, triple quadrupole mass spectrometry, MALDI-TOF mass    spectrometry, MALDI mass spectrometry, or time of flight mass    spectrometry, or any combination thereof-   8. The method of embodiment 7, wherein the mode of mass spectrometry    used is Selected Reaction Monitoring (SRM), Multiple Reaction    Monitoring (MRM), or multiple Selected Reaction Monitoring (mSRM),    or any combination thereof-   9. The method of any of embodiments 1 to 8, wherein the one or more    modified or unmodified ER, PR, and/or Ki67 fragment peptides    comprise two, three, four, five, six, seven, eight, nine or ten    different amino acid sequence independently selected from those set    forth as SEQ ID NO:1, SEQ ID NO:2, SEQ ID NO:3, SEQ ID NO:4, SEQ ID    NO:5, SEQ ID NO:6, SEQ ID NO:7, SEQ ID NO:8, SEQ ID NO:9, and SEQ ID    NO:10.-   10. The method of any of embodiments 1 to 9, wherein the biological    sample is a blood sample, a urine sample, a serum sample, an ascites    sample, a sputum sample, lymphatic fluid, a saliva sample, a cell,    or a solid tissue.-   11. The method of embodiment 10, wherein the tissue is formalin    fixed tissue.-   12. The method of embodiment 10 or 11, wherein the tissue is    paraffin embedded tissue.-   13. The method of embodiment 10, wherein the tissue is obtained from    a tumor.-   14. The method of embodiment 13, wherein the tumor is a primary    tumor.-   15. The method of embodiment 13, wherein the tumor is a secondary    tumor.-   16. The method of any of embodiments 1 to 15, further comprising    quantifying a modified or unmodified ER, PR, and/or Ki67 fragment    peptide.-   17. The method of embodiment 16, wherein quantifying a modified or    unmodified fragment peptide comprises comparing the amount of one or    more ER, PR, and/or Ki67 fragment peptides comprising an amino acid    sequence of about 8 to about 45 amino acid residues of ER, PR,    and/or Ki67 as shown in SEQ ID NO:1, SEQ ID NO:2, SEQ ID NO:3, SEQ    ID NO:4, SEQ ID NO:5, SEQ ID NO:6, SEQ ID NO:7, SEQ ID NO:8, SEQ ID    NO:9 and SEQ ID NO:10 in one biological sample to the amount of the    same ER, PR, and/or Ki67 fragment peptide in a different and    separate biological sample.-   18. The method of embodiment 17, wherein quantifying one or more    modified or unmodified ER, PR, and/or Ki67 fragment peptides    comprises determining the amount of each of said one or more ER, PR,    and/or Ki67 fragment peptides in a biological sample by comparison    to an added internal standard peptide of a known amount, wherein    each of the ER, PR, and/or Ki67 fragment peptides in the biological    sample is compared to an internal standard peptide having the same    amino acid sequence.-   19. The method of embodiment 18, wherein the internal standard    peptide is an isotopically labeled peptide.-   20. The method of embodiment 19, wherein the isotopically labeled    internal standard peptide comprises one or more heavy stable    isotopes selected from the group consisting of ¹⁸O, ¹⁷O, ³⁴S, ¹⁵N,    ¹³C, and ²H, or any combinations thereof.-   21. The method of any of embodiments 1 to 20, wherein detecting    and/or quantifying the amount of one or more modified or unmodified    ER, PR, and/or Ki67 fragment peptides in the protein digest    indicates the presence of modified or unmodified ER, PR, and/or Ki67    proteins and an association with cancer, and in particular breast    cancer, in the subject.-   22. The method of embodiment 21, further comprising correlating the    results of detecting and/or quantifying amounts of one or more    modified or unmodified ER, PR, and/or Ki67 fragment peptides, or the    amount of the ER, PR, and/or Ki67 proteins to the diagnostic    stage/grade/status of the cancer.-   23. The method of embodiment 22, wherein correlating the results of    detecting and/or quantifying the amount of one or more modified or    unmodified ER, PR, and/or Ki67 fragment peptides, or the amount of    the ER, PR, and/or Ki67 protein to the diagnostic stage/grade/status    of the cancer is combined with detected and/or quantified amounts of    other proteins, or peptides from other proteins, in a multiplex    format to provide additional information about the diagnostic    stage/grade/status of the cancer.-   24. The method of any one of embodiments 1 to 23, further comprising    selecting for the subject, from which the biological sample is    obtained, a treatment based on the presence, absence, or amount of    one or more ER, PR, and/or Ki67 fragment peptides or the amount of    ER, PR, and/or Ki67 proteins.-   25. The method of any one of embodiments 1 to 24, further comprising    administering to the patient, from which the biological sample is    obtained, a therapeutically effective amount of a therapeutic agent,    wherein the therapeutic agent and/or amount of the therapeutic agent    administered is based upon the amount of one or more modified or    unmodified ER, PR, and/or Ki67 fragment peptides or the amount of    ER, PR, and/or Ki67 proteins.-   26. The method of embodiments 24 or 25, wherein the treatment or the    therapeutic agent is directed to cancer cells expressing ER, PR,    and/or Ki67 proteins.-   27. The method of any of embodiments 1 to 26, wherein the biological    sample is formalin fixed tumor tissue that has been processed for    quantifying the amount of one or more modified or unmodified ER, PR,    and/or Ki67 fragment peptides employing the Liquid Tissue™ protocol    and reagents.-   28. The method of any of embodiments 1-27, wherein said one or more    modified or unmodified ER, PR, and/or Ki67 fragment peptides is one    or more, two or more, three or more, four or more, five or more, or    six or more of the peptides in Table 1.-   29. The method of any of embodiments 1-28, comprising quantifying    the amount of one, two, three, four, five, six, or seven of the    peptides in Table 2.-   30. A composition comprising one, two, three, four, five, six,    seven, eight, nine, or ten of the peptides in Table 1 or antibodies    thereto, said composition optionally excluding one, two, three,    four, five, or more peptides of ER, PR, and/or Ki67 that are not    peptides of SEQ ID NOs: 1, 2, 3, 4, 5, 6, 7, 8, 9, or 10.-   31. The composition of embodiment 30, comprising one, two, three,    four, five, six, or seven of the peptides of Table 2 or antibodies    thereto, said composition optionally excluding one, two, three,    four, five, or more peptides of ER, PR, and/or Ki67 that are not    peptides of SEQ ID NOs: 1, 2, 3, 4, 5, 6, or 7.

It is to be understood that the description, specific examples and data,while indicating exemplary aspects, are given by way of illustration andare not intended to limit the present disclosure. Various changes andmodifications within the present disclosure will become apparent to theskilled artisan from the discussion, detailed description and datacontained herein, and thus are considered part of the subject matter ofthis application.

1. A method for measuring the amount of the Ki67 protein in a biologicalsample of formalin-fixed tissue, comprising detecting and quantifyingthe amount of a Ki67 fragment peptide in a protein digest prepared fromsaid biological sample using mass spectrometry; and calculating theamount of Ki67 protein in said sample; wherein said Ki67 fragmentpeptide is the peptide of SEQ ID NO:6 or SEQ ID NO:7 and wherein theamount is a relative amount or an absolute amount.
 2. The method ofclaim 1, further comprising the step of fractionating the protein digestprior to detecting and quantifying the amount of said Ki67 fragmentpeptide. 3-4. (canceled)
 5. The method of claim 1, wherein the proteindigest comprises a protease digest.
 6. The method of claim 5, whereinthe protein digest comprises a trypsin digest. 7-11. (canceled)
 12. Themethod of claim 1, wherein the tissue is paraffin embedded tissue. 13.The method of claim 1, wherein the tissue is obtained from a tumor.14-16. (canceled)
 17. The method of claim 1, wherein quantifying saidfragment peptide comprises comparing the amount of said fragment peptidein one biological sample to the amount of the same Ki67 fragment peptidein a different and separate biological sample.
 18. The method of claim17, wherein quantifying said fragment peptide comprises determining theamount of said fragment peptide in a biological sample by comparison toan added internal standard peptide of a known amount, having the sameamino acid sequence. 19-20. (canceled)
 21. The method of claim 1,wherein detecting and quantifying the amount of said Ki67 fragmentpeptide in the protein digest indicates the presence of modified orunmodified Ki67 protein and an association with cancer in the subject.22. The method of claim 21, further comprising correlating the resultsof detecting and quantifying amounts of said Ki67 fragment peptide, orthe amount of the Ki67 protein to the diagnostic stage/grade/status ofthe cancer.
 23. The method of claim 22, wherein correlating the resultsof detecting and quantifying the amount of said Ki67 fragment peptide,or the amount of the Ki67 protein to the diagnostic stage/grade/statusof the cancer is combined with detected and/or quantified amounts ofother proteins, or peptides from other proteins, in a multiplex formatto provide additional information about the diagnosticstage/grade/status of the cancer.
 24. (canceled)
 25. The method of claim1, further comprising administering to the patient, from which thebiological sample is obtained, a therapeutically effective amount of atherapeutic agent, wherein the therapeutic agent and/or amount of thetherapeutic agent administered is based upon the amount of said Ki67fragment peptide or the amount of Ki67 protein.
 26. The method of claim25, wherein the treatment or the therapeutic agent is directed to cancercells expressing Ki67 protein. 27-31. (canceled)
 32. The method of claim1, further comprising detecting and/or quantifying the amount of atleast one additional fragment peptide selected from the group consistingof ER and PR fragment peptides in said protein digest using massspectrometry; and calculating the amount of ER and/or PR protein in saidsample; and wherein the amount is a relative amount or an absoluteamount
 33. The method of claim 33, wherein said additional fragmentpeptide is a ER fragment peptide.
 34. The method of claim 33, whereinsaid additional fragment peptide is a PR fragment peptide.
 35. Themethod of claim 1, wherein said fragment peptide is the peptide of SEQID NO:7.